PROJECT SUMMARY/ABSTRACT Hepatocyte injury and cell death underlie the development of hepatic failure and progression to the complications of fibrosis, cirrhosis and hepatocellular carcinoma in human liver disease. Despite recent advances, the mechanisms of hepatocyte injury and death remain poorly understood. Liver injury results primarily from the effects of factors generated by the excessive sterile inflammatory response that accompanies hepatocyte injury and death. Over the previous funding period we demonstrated that conditions that predispose to liver injury are associated with decreased function of the lysosomal degradative pathway of autophagy in both hepatocytes and hepatic macrophages. We and others have also shown that autophagy functions to prevent liver injury and inflammation, but the mechanisms of autophagy's protective effects remain unclear. The objective of this proposal is to delineate the mechanisms by which the impairment in autophagy in hepatocytes and macrophages promotes the development of liver injury and inflammation. Preliminary studies indicate that macrophages and hepatocytes with decreased autophagy have heightened inflammatory responses to toll-like receptor 2 (TLR2) stimulation that causes liver injury. In addition, hepatocytes with impaired autophagy become sensitized to proinflammatory cytokine toxicity from the synergistic effects of IL-1? and TNF, and release increased amounts of proinflammatory damage-associated molecular patterns (DAMPs). Based on these and other preliminary findings, the central hypothesis of this proposal is that decreased autophagy amplifies the proinflammatory activation of macrophages and hepatocytes through effects on specific cellular signaling pathways to heighten the sterile inflammatory response and promote hepatocyte death in liver injury. We will test this hypothesis through investigations contained in three Specific Aims that will examine liver injury in cell culture and mouse models with a genetic decrease in autophagy. First, we will test the hypothesis that impaired autophagy promotes liver injury by hyperactivation of a TLR2-dependent innate immune response in both macrophages and hepatocytes through the mechanism of a docking protein 1-mediated decrease in extracellular signal-regulated kinase 1/2 signaling. Second, we will test the hypothesis that with decreased autophagy the proinflammatory cytokines IL-1? and TNF induce synergistic lysosomal oxidant stress that triggers cathepsin-dependent hepatocyte necrosis. Third, we will test the hypothesis that with decreased autophagy dying hepatocytes induce a local and systemic innate immune response through the secretion of exosomes containing DAMPs that are normally sequestered and degraded by autophagy. The ultimate goal of these investigations is to understand how an impairment in autophagy promotes liver disease progression through increases in the innate immune response and hepatocyte death in order to develop new treatment modalities for human liver disease.